Coupling of vinculin to F-actin demands Syndecan-4 proteoglycan

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in orderto address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior. (C) 2016 Elsevier B.V. All rights reserved.CAPES (Coordenagdo de Aperfeicoamento de Pessoal de Nivel Superior)CNPq (Conselho Nacional de Desenvolvimento Cientffico e Tecnologico)FAPESP (Fundacao de Amparo a Pesquisa do Estado de sao Paulo), BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Sao Paulo, SP, BrazilUniv Liverpool, Inst Integrat Biol, Dept Biochem, Liverpool, Merseyside, EnglandUniv Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniv Houston, Coll Optometry, TOSI, Houston, TX USAUniv Fed Sao Paulo, Grp Interdisciplinar Ciencias Exatas Saude, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Diadema, SP, BrazilUniv Fed Sao Paulo, Grp Interdisciplinar Ciencias Exatas Saude, Sao Paulo, SP, BrazilFAPESP: 15/08782-3FAPESP: 15/03964-6Web of Scienc

Linking Employee Stakeholders to Environmental Performance: The Role of Proactive Environmental Strategies and Shared Vision

Drawing on the natural-resource-based view (NRBV), we propose that employee stakeholder integration is linked to environmental performance through firms’ proactive environmental strategies, and that this link is contingent on shared vision. We tested our model with a cross-country and multi-industry sample. In support of our theory, results revealed that firms’ proactive environmental strategies translated employee stakeholder integration into environmental performance. This relationship was pronounced for high levels of shared vision. Our findings demonstrate that shared vision represents a key condition for advancing the corporate greening agenda through proactive environmental strategies. We discuss implications for the CSR and the environmental management literatures, with a particular focus on the NRBV and stakeholder integration debates

Keratan sulphate in the tumour environment

Keratan sulphate (KS) is a bioactive glycosaminoglycan (GAG) of some complexity composed of the repeat disaccharide D-galactose β1→4 glycosidically linked to N-acetyl glucosamine. During the biosynthesis of KS, a family of glycosyltransferase and sulphotransferase enzymes act sequentially and in a coordinated fashion to add D-galactose (D-Gal) then N-acetyl glucosamine (GlcNAc) to a GlcNAc acceptor residue at the reducing terminus of a nascent KS chain to effect chain elongation. D-Gal and GlcNAc can both undergo sulphation at C6 but this occurs more frequently on GlcNAc than D-Gal. Sulphation along the developing KS chain is not uniform and contains regions of variable length where no sulphation occurs, regions which are monosulphated mainly on GlcNAc and further regions of high sulphation where both of the repeat disaccharides are sulphated. Each of these respective regions in the KS chain can be of variable length leading to KS complexity in terms of chain length and charge localization along the KS chain. Like other GAGs, it is these variably sulphated regions in KS which define its interactive properties with ligands such as growth factors, morphogens and cytokines and which determine the functional properties of tissues containing KS. Further adding to KS complexity is the identification of three different linkage structures in KS to asparagine (N-linked) or to threonine or serine residues (O-linked) in proteoglycan core proteins which has allowed the categorization of KS into three types, namely KS-I (corneal KS, N-linked), KS-II (skeletal KS, O-linked) or KS-III (brain KS, O-linked). KS-I to -III are also subject to variable addition of L-fucose and sialic acid groups. Furthermore, the GlcNAc residues of some members of the mucin-like glycoprotein family can also act as acceptor molecules for the addition of D-Gal and GlcNAc residues which can also be sulphated leading to small low sulphation glycoforms of KS. These differ from the more heavily sulphated KS chains found on proteoglycans. Like other GAGs, KS has evolved molecular recognition and information transfer properties over hundreds of millions of years of vertebrate and invertebrate evolution which equips them with cell mediatory properties in normal cellular processes and in aberrant pathological situations such as in tumourogenesis. Two KS-proteoglycans in particular, podocalyxin and lumican, are cell membrane, intracellular or stromal tissue–associated components with roles in the promotion or regulation of tumour development, mucin-like KS glycoproteins may also contribute to tumourogenesis. A greater understanding of the biology of KS may allow better methodology to be developed to more effectively combat tumourogenic processes

Hyaluronan derived from the limbus is a key regulator of corneal lymphangiogenesis

Purpose: We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix. Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea. Methods: To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis. Results: Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury. Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice. Conclusion: These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation

Hyaluronan derived from the limbus is a key Regulator of Corneal Lymphangiogenesis

Purpose: Corneal lymphangiogenesis and angiogenesis leads to the loss of corneal transparency. We have recently shown that in the cornea hyaluronan (HA) is present primarily in the limbal region and plays a key role regulating the limbal stem cell phenotype. Given the HA receptor LYVE-1 is highly expressed in corneal lymphatic vessels we investigated whether HA could play a role in regulating corneal lymphangiogenesis. Methods: Wild-type (wt) and hyaluronan synthase (HAS) knockout mice - specifically combined Has1-/- and Has3 -/- null mice (HAS1-/-;HAS3-/-) and conditional Has2 knock-out mice (HAS2D/DCorEpi), were used. The mice were subjected to injury, alkali burn or suture placement, to investigate the role of HA on corneal lymphangiogenesis. Corneal buttons were also obtained from different developmental time-points to study the role of HA in lymphatic vessel development. The corneas were analyzed by whole mount immunohistochemistry and entire corneas were imaged under an LSM 800 confocal microscope using the both the z-stack and tiling mode. Primary lymphatic vessel endothelial cells from human dermis (hDLECs) and lymph node (hLLECs) were used for tube formation assay and cell proliferation assay in vitro. Results: After injury both wild-type and HAS1-/-;HAS3-/- mice presented both an increase in HA expression and lymphangiogenesis. Interestingly, lymphatic vessels extended exclusively into HA rich areas. In stark contrast, HAS2D/DCorEpi mice did not upregulate HA synthesis after injury and, in turn, did not present lymphangiogenesis. Our developmental studies revealed first HA is expressed in the corneal limbus and thereafter lymphatic vessels invade this region. Our in vitro studies corroborated our in vivo data, with both HA increasing the proliferation and tube formation ability of hDLECs and hLLECs. Conclusions: HA regulates corneal lymphangiogenesis, both during development and after injury. These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis could be used to reduce corneal scarring and also prevent rejection after corneal transplantation

Hyaluronan derived from the limbus is a key regulator of corneal lymphangiogenesis

Purpose: We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix. Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea. Methods: To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis. Results: Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury. Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice. Conclusion: These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation

"GAG-ing with the neuron": The role of glycosaminoglycan patterning in the central nervous system.

Proteoglycans (PGs) are a diverse family of proteins that consist of one or more glycosaminoglycan (GAG) chains, covalently linked to a core protein. PGs are major components of the extracellular matrix (ECM) and play critical roles in development, normal function and damage-response of the central nervous system (CNS). GAGs are classified based on their disaccharide subunits, into the following major groups: chondroitin sulfate (CS), heparan sulfate (HS), heparin (HEP), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA). All except HA are modified by sulfation, giving GAG chains specific charged structures and binding properties. While significant neuroscience research has focused on the role of one PG family member, chondroitin sulfate proteoglycan (CSPG), there is ample evidence in support of a role for the other PGs in regulating CNS function in normal and pathological conditions. This review discusses the role of all the identified PG family members (CS, HS, HEP, DS, KS and HA) in normal CNS function and in the context of pathology. Understanding the pleiotropic roles of these molecules in the CNS may open the door to novel therapeutic strategies for a number of neurological conditions

Differences in the expression of glycosaminoglycans in human fibroblasts derived from gingival overgrowths is related to TGF-beta up-regulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-beta 1 and TGF-beta type 11 receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in alpha-L-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-beta over-expression.2812433Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Jairo Ramos postdoctoral fellowshipFAP UNIFESP (Fundacao de Apoio a Pesquisa)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES